Deparia × nanakuraensis K.Hori (Athyriaceae), a new hybrid pteridophyte from Japan

A peer-reviewed open-access journal

PhytoKeys 165: 69-84 (2020)

&
ie cee $¢PhytoKeys

https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research

Deparia xnanakuraensis K.Hori (Athyriaceae),
a new hybrid pteridophyte from Japan

Kiyotaka Hori!

| The Kochi prefectural Makino Botanical Garden, Kochi, Japan

Corresponding author: Kiyotaka Hori (khori@makino.or.jp)

Academic editor: Th. Almeida | Received 20 August 2020 | Accepted 6 October 2020 | Published 28 October 2020

Citation: Hori K (2020) Deparia xnanakuraensis K.Hori (Athyriaceae), a new hybrid pteridophyte from Japan.
PhytoKeys 165: 69-84. https://doi.org/10.3897/phytokeys.165.57837

Abstract

I describe Deparia xnanakuraensis hyb. nov. and discuss differences in morphological characteristics be-
tween parental species D. pterorachis and D. viridifrons with chromosome counting, plastid, and nuclear
DNA markers. The new hybrid is endemic to the eastern and northern parts of Japan. Based on the criteria
of the International Union for Conservation of Nature and Natural Resources, this new species is here

considered Data Deficient. The ploidy level is diploid sterile.

Keywords
Athyriaceae, Deparia, new hybrid, Japan

Introduction

The genus Deparia Hook. & Grev. is one of the largest groups in the Athyriaceae
family. It contains 60-90 species mostly in East Asia, with some species distributed
in Africa, western Indian Ocean, northeastern North America, the Hawaiian Islands,
Australia, New Zealand, and South Pacific Islands (Kato 1984; Rothfels et al. 2012; He
et al. 2013; Kuo et al. 2016, 2018; PPG I 2016; Moran et al. 2019).

The genus is characterized by hair-like scales and disconnected grooves between
rachises and costae (Kato 1973, 1977, 1984; Rothfels et al. 2012; Sundue and Roth-
fels 2014; Kuo et al. 2018). These two features have not been observed in the genera
Anisocampium C.Presl, Athyrium Roth., Diplazium Sw., Ephemeropteris R.C.Moran &

Sundue, and Pseudathyrium Newman but in some species in the Athyriaceae family

Copyright Kiyotaka Hori. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

70 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

(Kato 1973; Rothfels et al. 2012; Moran et al. 2019). In addition, narrowly U-shaped
rachis grooves are also a unique character of the genus Deparia (Kuo et al. 2018). The
basic chromosome number of Deparia is 40, contrary to Diplazium of 41 (Sano et al.
2000; Rothfels et al. 2012).

In Japan, several hybrids of the genus Deparia have been described: D. x birii Fraser-
Jenk. (Fraser-Jenkins 2008), D. xkiyozumiana (Sa.Kurata) Y.Shimura (Shimura 1980),
pentaploid sterile D. dancea (Thunb.) Fraser-Jenk. (Nakato and Mitui 1979), D. xJo-
batocrenata (Tagawa) M.Kato (Kato 1984; Ebihara 2017), D. xmusashiensis (H.Ohba)
Seriz. (Serizawa 1981), pentaploid sterile D. petersenii (Kunze) M.Kato (Shinohara
et al. 2003), D. xtogakushiensis Otsuka & Fujiw. (Otsuka and Fujiwara 1999), and
D. xtomitaroana (Masam.) R.Sano (Sano et al. 2000). Furthermore, Ebihara (2017)
mentioned several combinations of hybrids that are not still described.

The Deparia okuboana complex (Athyriaceae) is recently defined by Ebihara
(2017) as consisting of D. okuboana (Makino) M.Kato (apogamous triploid; Hira-
bayashi 1970), D. coreana (Christ) M.Kato (sexual tetraploid, Nakato and Ebihara
2018), D. henryi (Baker) M.Kato (apogamous triploid, Nakato and Ebihara 2018), D.
viridifrons (Makino) M.Kato (sexual diploid; Hirabayashi 1970), D. unifurcata (Bak-
er) M.Kato (apogamous triploid; Hirabayashi 1970), D. pterorachis (Christ) M.Kato
(sexual diploid; Hirabayashi 1970). There is continuous morphological variation be-
tween D. coreana, D. henryi, D. okuboana, and D. unifurcata (Ebihara 2017). Kuo et
al. (2018) identified that these members belong to sect. Dryoathyrium. Hori (2018)
reported there were reticulate relationships in the D. okuboana complex with sect. Lu-
nathyrium (Kuo et al. 2018) based on plastid and nuclear DNA marker. In addition,
Ebihara (2017) mentioned undescribed diploid sterile hybrid between D. pterorachis
and D. viridifrons based on morphology and ploidy level. This study described this new
hybrid of D. pterorachis and D. viridifrons, Deparia xnanakuraensis K.Hori, based on
morphological characteristics, chromosome number, plastid, and nuclear DNA marker.

Materials and methods

Plant materials, Chromosome count, and DNA extraction

In this study, Deparia viridifrons, D. xnanakuraensis, and D. pterorachis were
investigated in molecular DNA analysis. Other members of the D. okuboana complex
(D. coreana, D. henryi, D. okuboana, D. unifurcata) and Japanese members of the sect.
Lunathyrium (D. pycnosora var. albosquamata M.Kato, D. pycnosora (Christ) M.Kato
var. pycnosora, D. pycnosora var. mucilagina M.Kato) were also used as materials.
Voucher information for all samples is listed in Appendix I. All voucher specimens
have been deposited in the Makino Herbarium of Tokyo Metropolitan University
(MAK), and/or the Kochi Prefectural Makino Botanical Garden (MBK). The DNA

sequences of Athyrium melanolepis Christ, A. crenulatoserrulatum Makino, A. opacum

Depariaxnanakuraensis K.Hori (Athyriaceae) FA

Copel., Diplazium chinense (Baker) C.Chr., Di. esculentum (Retz.) Sw., Di. wichurae
(Mett.) Diels were used as outgroups, quoted from the Genbank database.

Additionally, specimens from the Collection Database and Materials of TNS
(http://db.kahaku.go.jp/webmuseum/), PE (http://pe.ibcas.ac.cn/en/), TAIF (http://
taif.tfri.gov.tw/search.php), and from the JSTOR Global Plants (https://plants.jstor.
org/) as well as from the Global Biodiversity Information Facility (GBIF: https://www.
gbif.org) database were checked.

For the conservation assessment, the area of occupancy (AOO) and extent of oc-
currence (EOO) were estimated using GeoCAT (Bachman et al. 2011), default set-
tings for grid size were applied. In addition, mitotic chromosomes from D. xnanaku-
raensis were counted.

To observe mitotic chromosomes, root tips were collected in the field, and pre-
treated with 0.004 M 8-hydroxyquinoline for 6 h at approximately 17—20 °C. After
fixation in ethanol and acetic acid (3:1) for 15-30 min, the root tips were hydrolyzed
in 1 N HCl at 60 °C for 1-3 min and then squashed in 2% aceto-orcein solution. The
chromosomes were observed under a microscope (Leica DM2500) and then photo-
graphed by using a digital camera (Leica MC170 HD).

For the molecular analyses, total DNA was extracted from silica-dried leaves using
cetyltrimethylammonium bromide solution, according to Doyle and Doyle (1990).

Plastid and nuclear DNA sequencing

trnL-F was used as the maternally-inherited (Gastony and Yatskievych 1992; Kuo et
al. 2018) plastid DNA marker (F: 5'-ATTTGAACTGGTGACACGAG-3' and FernL
1 Irl: 5'-GGYAATCCTGAGCAAATC-3'; Taberlet et al. 1991; Li et al. 2009). AKI
(AK4F: 5'-GATGAAGCCATCAAGAAACCA-3' and AKR2: 5'-ATGGATCCAGC-
GACCAGTAA-3'; Hori and Murakami 2019) was used as a biparentally-inherited nu-
clear marker for polymerase chain reaction-single-strand conformation polymorphism
(PCR-SSCP) analysis, which was used to determine allelic variation in each individual
(Hori and Murakami 2019).

PCR amplification was performed using PrimeSTAR Max DNA Polymerase (Ta-
kara, Kyoto, Japan). PCR entailed an initial denaturation step at 95 °C for 10 min,
followed by 35 cycles of denaturation, annealing, and elongation steps at 98 °C for
10 s, 55 °C for 5 s, and 72 °C for 5 s, respectively, using a Model 9700 thermal cycler
(Applied Biosystems, Foster City, CA, USA).

Gel electrophoresis of AKI PCR products was performed using gels of 5|0% MDE
gel solution (Lonza) containing 2% glycerol at 15 °C for 16 h at 300 V, followed by
silver staining. For sequencing of the bands separated on the gels, the polyacrylamide
gel was dried after silver staining by sandwiching the gel between Kent paper and a
cellophane sheet on an acrylic backplate at 55 °C for 4 h. To extract the DNA, a piece
of the DNA band was peeled from the dried gel using a cutter knife and incubated
in 50 uL of Tris-EDTA buffer (10-mM Tris-HCl and 1-mM EDTA, pH 8.0) at 4 °C

2 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

overnight. The supernatant solution was used as a template for further PCR amplifica-
tion with the same primer set employed for initial PCR amplification.

PCR products were purified using Illustra ExoStar 1-Step (GE Healthcare, Wis-
consin, USA) and used as templates for direct sequencing. Reaction mixtures for se-
quencing were prepared using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Ap-
plied Biosystems). The reaction mixtures were analyzed using an ABI 3130 Genetic

Analyzer (Applied Biosystems).

Molecular analysis

The accession numbers of DNA sequences in the datasets were shown in Appendix I.
The sequences were aligned using MUSCLE (Edgar 2004) and assessed with Bayesian
inference (BI) analysis using MrBayes 3.2.6 (Ronquist et al. 2012), maximum par-
simony (MP), and maximum likelihood (ML) analysis using the MEGA X software
(Kumar et al. 2018). Indels were treated as missing characters in all analyses. In the
BI analysis, the best-fit model (trnZ-F: HKY+I model; AK7: HKY model) of sequence
evolution for each DNA region was selected using jModelTest 2.1.10 (Darriba et al.
2012). Four Markov chain Monte Carlo chains were run simultaneously and sampled
every 100 generations for 1 million generations in total. Tracer 1.7.1 (Rambaut et al.
2018) was used to examine the posterior distribution of all parameters and their associ-
ated statistics, including estimated sample sizes. ‘The first 2,500 sample trees from each
run were discarded as burn-in periods. The MP tree was obtained using the Tree-Bi-
section-Regrafting (TBR) algorithm (Nei and Kumar 2000) at search level 3, at which
the initial trees were obtained by the random addition of sequences (100 replicates).
The confidence level of the monophyletic groups was estimated with 1,000 MP boot-
strap pseudo-replicates. In ML analysis, the best-fitting model of sequence evolution
for each marker was selected using MEGA; Tamura 3-parameter + I model was used
for trnL-F and HKY model for AK/. Initial trees for the heuristic search were obtained
automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood approach and then
selecting the topology with superior log likelihood value. The bootstrap method with
1,000 replications was employed to estimate the confidence levels of monophyletic

groups in MP and ML analysis.

Results

Chromosome count

Mitotic metaphase chromosome number observed in an individual of D. xnanaku-
raensis (Hori 3391) was 2n = 80 (Figure 1). This individual had shrunken sporan-
gium with no spores. The basic chromosome numbers of the genus Deparia is x=
40 (Sano et al. 2000; Rothfels et al. 2012), and suitably, this sample was found to
be a sterile diploid.

Depariaxnanakuraensis K.Hori (Athyriaceae) 73

Figure |. Photograph and sketch of mitotic metaphase chromosomes (27 = 80) of D. x nanakuraensis
(Hori 3391).

Plastid and nuclear DNA phylogenetic trees

We sequenced 653-746 bp of the zrnL-F intergenic spacer from different specimens.
The aligned trnL-F matrix was 765 bp, of which 121 characters (15%) were parsi-
mony-informative. For the AK/ intron, we sequenced 338-590 bp of the intron for
each specimen, yielding a 604 bp aligned matrix, of which 74 characters (12%) were
parsimony-informative.

The ML trees according to the sequences of trnL-F (In L = -2309.05) and AKI
(In Z = -1616.59) with bootstrap percentages (BPs), Bayesian posterior probabilities
(PP) were shown in Figures 2, 3, respectively. In the trnL-F phylogeny, the haplo-
type of D. pterorachis and D. viridifrons composed different clades with D. coreana,
D. henryi, and D. okuboana which were supported by BP (>70) and PP (>0.90)
values. In the AK/ phylogeny, the two clades containing D. pterorachis and D. vir-
idifrons were supported by BP, but D. viridifrons was not supported by PP value.
Deparia xnanakuraensis had the same haplotype of D. pterorachis and D. viridifrons
in both trnL-F and AKI phylogenies. Other members of the D. okuboana complex
(D. coreana, D. henryi, D. okuboana, D. unifurcata) shared the same alleles with D.
viridifrons partly (Hori 2018), but the combination of alleles was different from D.
xnanakuraensis. Japanese members of the sect. Lunathyrium (D. pycnosora var. albos-
guamata, D. pycnosora vat. pycnosora, D. pycnosora var. mucilagina) did not share any
alleles with D. xnanakuraensis. Therefore, D. xnanakuraensis can be of origin hybrid
from D. pterorachis and D. viridifrons.

Taxonomic treatment

Deparia xnanakuraensis K.Hori, hyb. nov.
urn:lsid:ipni.org:names:7721257 1-1
Figure 4

Type. Japan. Honshu: Akita prefecture, Noshiro city, Futatsui town, Nanakura-shrine,
40°12'9.48"N, 140°15'29.82"E, alt. 23 m, deciduous forest containing Acer miyabei

74 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

D. viridifrons Hori2971
D. viridifrons Hori3060

100/99/1 D. viridifrons Hori3061

D. henryi Hori3028

79/-/- D. okuboana Hori3033

D. coreana Hori3047

L_____-——. unifurcata Hori3029
91/83/-

D. pterorachis Hori3053

D. pterorachis Hori3054

100/99/0.99
ae D. pterorachis Hori3055

D. x nanakuraensis Hori3391

100/100/1 D. x nanakuraensis Hori3392

D. x nanakuraensis Hori3393

__—— D. pycnosora var. albosquamata Hori3382

_— D. pycnosora var. mucilagina Hori3380

98/99/1
99/99/1 ‘— D. pycnosora var. pycnosora Hori3052

Diplazium wichurae LC468190*

98/97/1
Di. chinense LC468193*
Athyrium melanolepis MG183541*
100/100/0.99 A. crenulatoserrulatum MG183578
100/99/1 ——_ A. opacum KX656057*
0.020

Figure 2. The ML tree based on the sequence variation of the gene tnL-F (In L = -2309.05) with PP
(>0.90) and BP (+70) of ML/MP/BI analyses on each branch. The sequences with asterisks were quoted
from Genbank.

Maxim., Aesculus turbinata Blume, Cercidiphyllum japonicum Siebold & Zucc., Cryp-
tomeria japonica (Thunb. ex L.f.) D.Don, Dryopteris monticola (Makino) C.Chr., and
Pachysandra terminalis Siebold & Zucc., on soil, 7 Jul 2020, K. Hori 3391 (holotype:
MAK467056; isotype: MBK).

Description. Terrestrial, summer green fern. Rhizomes creeping, occasionally
branched, with buds, stramineous, 15—25 x 4-7 cm, closely set with roots and persis-

Depariaxnanakuraensis K.Hori (Athyriaceae) vay

. x nanakuraensis Hori3391
. x nanakuraensis Hori3392
. x nanakuraensis Hori3393
. viridifrons Hori2971

D
D
D
D
88/88)! 5 viridifrons Hori3060
D
D
D
D

. Viridifrons Hori3061

. coreana Hori3047
- /84 /-

. okuboana Hori3033

. henryi Hori3028

r— D. unifurcata Hori3029-allele1
D. unifurcata Hori3029-allele2

a

D. henryi Hori3028

D. okuboana Hori3033

; _— D. pycnosora var. mucilagina Hori3380
77/82/0.90 D. pterorachis Hori3053

D. pterorachis Hori3054
99/99/1 | D. pterorachis Hori3055

82/87/0.97

91/96/0.92
D. x nanakuraensis Hori3391

D. x nanakuraensis Hori3392

99/99/1 -/74/0.99 D. x nanakuraensis Hori3393

ae D. pycnosora var. albosquamata Hori3382-allele1
| D. coreana Hori3047

= »— D. pycnosora var. pycnosora Hori3052-allele1

-/72/0.94 D. pycnosora var. pycnosora Hori3052-allele2

D. henryi Hori3028

97/93/1 '—— D. pycnosora var. albosquamata Hori3382-allele2
Athyrium melanolepis MN267422*

;_— A. crenulatoserrulatum LC421516*

A. opacum MN267421*
———— A, decurrentialatum LC421512*

Diplazium esculentum LC468186*
91/87/0.99 Di. wichurae LC468187*

100/100/1 ~— Di. wichurae LC468188*

98/99/1

99/99/1

} |
0.010

Figure 3. The ML tree based on the sequence variation of the gene AK/ (In L = -1616.59) with PP
(>0.90) and BP (+70) of ML/MP/BI analyses on each branch. The sequences with asterisks were quoted
from Genbank.

tent, densely clothed by old stipe bases, glabrous; fronds 4-6 per rhizome; stipes whit-
ish green, 30-40 x 0.8—1.5 cm, sparsely clothed with stramineous scales at the base
(1-1.5 x 0.5-1 cm), ovate; blades yellowish green adaxially, 3-pinnate-pinnatifid at the

76 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

Cop el ef

Figure 4. D. xnanakuraensis K.Hori A lower stipe scale B upper stipe scale C abaxial surface of frond

and stipe D detail of adaxial pinnule E detail of abaxial pinnule, and F rhizome and base of stipes.

A-F from the holotype (MAK467056) (illustration by K. Hori).

base, in the middle to upper section, 2-pinnate at the apex, 40-70 x 30-40 cm, del-
toid; rachises whitish green, glabrous, sparsely clothed with stramineous scales (2—5 x
1—2 mm) and black hairs adaxially; pinnae 10-15 pairs, ascending, lanceolate, shrunk-
en at base, alternate, petiolated (2-5 mm), sessile near the apex, lowest pinnae slightly
reduced, second lowest pair usually the largest, 25-30 x 4-8 cm; pinnules, alternate
on the basal and middle sections of the blade, 20-30 pairs on the basal and middle
sections of the blade, 15-20 pairs on the apex of the blade, reduced distally, lanceolate,
deeply serrated, vein-free, close to or reaching to the margin, 10-15 pairs in the middle
lobe; sori brown, tending to appear on the abaxial surface of the middle or upper part
of blades, oblong- to J-shaped, 1.5—3 mm long, on the apex or middle of veinlets, 5—10
per ultimate segment, persistent; izdusium entire to serrated on margin, sporangium
shrunken, spores absent.

Depariaxnanakuraensis K.Hori (Athyriaceae) ra

190 195 140 (7) aa
ae ane
te CS

140 145
Figure 5. Map showing the known distribution of D. xnanakuraensis in Japan. Red star indicates type
locality, other circles indicate examined specimens.

Etymology. The name derives from Nanakura-shrine, Futasui town, Noshiro City,
Akita prefecture, northeast Japan, where Deparia xnanakuraensis was first found.

Specimens examined. Japan. Honshu: Akita pref., Noshiro city, Futatsui town,
Nanakura-shrine, 40°12'9.48"N, 140°15'29.82"E, alt. 23 m, 7 Jul 2020, K. Hori 3392,
loc. cit., K. Hori 3393, loc. cit., K. Hori 3394, loc. cit., 10 Jul 2012, Y. Horii 35548 (TNS
01167830), loc. cit., Y Horii 35549 (TNS 01167829); Aomori pref., Hachinohe city,
Same town, Kamikoswa, alt. 100 m, 23 Aug 1975, coll. MZ. Neichi (TNS 1170337,
image!); /oc. cit., Kitsunetai, alt. 30m, 9 Jul 2005, coll. M. Neichi (TNS 01183638, im-
age!); Iwate pref., lwaizumi town, Atsuka, Matsugasawa, alt. 350 m, 18 Jul 1981, coll.
M. Neichi (TNS 01161869, image!); loc. cit., Ichinoseki city, Higashiyama cho, Naga-
saka, Nagahira, alt. 180 m, 22 Aug 1987, coll. MZ. Suzuki (TNS 932028, image!); loc.
cit., Maikawa, Ohira, alt. 120 m, 22 Sep 1986, coll. M. Suzuki (TNS 9320284image!);
Miyagi pref., Ishinomaki city, Mano, Uchihara, alt. 70 m, 25 May 1990, coll. K. Shogo
(TNS01184195, image!); loc. cit., Sendai city, Akiu town, Baba, alt. 200 m, 15 Oct
1983, coll. K. Shogo (TNS01184194, image!); Yamagata pref., Kamiyama city, Takano,
alt. 250 m, 5 Jun 1983, coll. N. Sakawa (TNS01161877, image!); Fukushima pref.,
Minamiaizu county, Shimosato town, Yunokami, alt. 500 m, 8 Sep 1972, coll. 77 Waku
(TNS01161873, image!); Saitama pref., Hannnou city, Kasasugitouge, alt. 500 m, 21

78 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

Figure 7. Juvenile of D. xnanakuraensis.

Depariaxnanakuraensis K.Hori (Athyriaceae) 7

5cm

Figure 8. Indefinite growth through bud (red arrow) on rhizome of D. xnanakuraensis.

Figure 9. Abaxial surface of pinnule and sori of A D. viridifrons B D. xnanakuraensis, and C D. ptero-
rachis (illustration by K. Hori).

Aug 1984, coll. 77 Iwata (TNS01140142, image!); loc. cit., 14 Sep 1980, coll. Y Kod-
ayashi (MBK0233005); loc. cit., 14 June 1981, coll. Y Kobayashi (MBK0232983).
Distribution and ecology. Deparia xnanakuraensis is known from the eastern and
northern part of Honshu in Japan (Figure 5). It was observed to grow on soil under de-
ciduous forest (Figure 6) or planted coniferous forest containing Cryptomeria japonica.
This hybrid is endemic to Japan. In the type locality, this hybrid comprised a popula-

tion of over 30 individuals with juveniles (Figure 7) although parents of D. viridifrons

80 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

and D. pterorachis were both absent, and sporangium had no spores. However, it is
expected that Deparia xnanakuraensis can reproduce young individuals from buds on
its rhizome (Figure 8).

Conservation status. [UCN Red List Category. Based on estimates from Geo-
CAT, the EOO of D. xnanakuraensis was 46,321 km?. The known AOO of D. xnana-
kuraensis was 44 km’. The localities correspond to less than 20 points, but I could not
check the population size on each locality. Therefore, available information is inad-
equate to support the assessment of its extinction risk. According to the IUCN (2012)
criteria, the category of Data Deficient (DD) is appropriate.

Discussion

Deparia xnanakuraensis presents almost intermediate morphologies between D. viridi-
frons and D. pterorachis species. Deparia viridifrons is characterized by having deltoid-
ovate or ovate-lanceolate fronds, reniform to U-shaped sori, pinnules with costal wing,
rounded serration of pinnules, and acute apex of pinnules. In contrast, D. pterorachis
has oblong fronds, oblong to J-shaped sori, pinnules truncated to costa; truncate serra-
tion of pinnules, and obtuse apex of pinnules (He et al. 2013; Ebihara 2017). Deparia
xnanakuraensis has deltoid fronds, oblong to J-shaped sori, pinnules with narrow costal
wing, rather rounded serration of pinnules, and a rather acute apex of pinnules (Fig-
ure 9, Table 1).

Kuo et al. (2018) classified D. viridifrons and D. pterorachis as the members of sect.
Dryoathyrium because lateral pinnules are not auricled, and these are closely related in
plastid DNA phylogeny (Kuo et al. 2018). Therefore, Deparia xnanakuraensis is infra
section hybrid in the sect. Dryoathyrium.

The ploidy level of this hybrid is the same as its parents because D. viridifrons and
D. pterorachis are both sexual diploid (Kurita 1963; Mitui 1966, 1968, 1970; Hira-
bayashi 1970). In addition, this can be the first report of a diploid sterile hybrid of the
genus Deparia from Japan although several hybrids have been described (Ebihara 2017).

In conclusion, this study described Deparia xnanakuraensis based on morphology,
cytology, and molecular DNA analysis. The morphological characteristics were inter-
mediate between its parents D. viridifrons and D. pterorachis. This hybrid can produce
young individuals from buds on its rhizome. Based on the criteria of the International
Union for Conservation of Nature and Natural Resources, this new species is here

Table |. Morphological comparison among D. xnanakuraensis and related species.

Characteristics | Shape of frond Shape of sori Margin of Base of pinnule | Serration of Apex of
D. viridifrons deltoid-ovate or | reniform to U-shaped serrated with costal wing rounded acute
D. xnanakuraensis deltoid oblong to J-shaped | entire to serrated | with narrow costal} rather rounded | rather acute
[ii jee i

D. pterorachis oblong oblong to J-shaped truncated to costa obtuse

Depariaxnanakuraensis K.Hori (Athyriaceae) 81

considered Data Deficient. This hybrid can be the first report of diploid sterile hybrid
of the genus Deparia from Japan. In future studies, it is expected that more hybrids of
the genus Deparia will be discovered and described from Japan.

Acknowledgments

I am grateful to Mr. Y. Horii for plant collections. This study was supported by a
Grant-in-Aid for JSPS Fellows 18K14785 to K. H.

References

Bachman S, Moat J, Hill A, de la Torre J, Scott B (2011) Supporting Red List threat assess-
ments with GeoCAT: Geospatial conservation assessment tool. ZooKeys 150: 117-126.
https://doi.org/10.3897/zookeys. 150.2109

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heu-
ristics and parallel computing. Nature Methods 9(8): 1-772. https://doi.org/10.1038/
nmeth.2109

Doyle JA, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus (San Francisco,
Calif) <13—1'S3

Ebihara A (2017) The standard of ferns and lycophytes in Japan 2. Gakken Plus, Tokyo. [in
Japanese]

Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Research 32(5): 1792-1797. https://doi.org/10.1093/nar/
ekh340

Fraser-Jenkins RC (2008) Deparia x birii. In: Fraser-Jeknkins RC (Ed.) Taxonomic Revision of
Three Hundred Indian Subcontinental Pteridophytes With a Revised Census-List. Bishen
Singh Mahendra Pal Singh, Dehradun, 249 pp.

Gastony GJ, Yatskievych G (1992) Maternal inheritance of the chloroplast and mitochondrial
genomes in cheilanthoid ferns. American Journal of Botany 79(6): 716-722. https://doi.
org/10.1002/}.1537-2197.1992.tb14613.x

He Z, Wang Z, Kato M (2013) Deparia. In: Wu ZY, Raven PH, Hong DY (Eds) Flora of Chi-
na 2-3: (Pteridophytes). Science Press, Beijing & Missouri Botanical Garden, St. Louis,
443-447,

Hirabayashi H (1970) Chromosome numbers in several species of Aspidiaceae (2). Shokubutsu
Kenkyu Zasshi 45: 45-52.

Hori K (2018) Hybrid origin of some species in the Deparia okuboana complex (Athyriaceae,
Polypodiidae) verified with DNA analysis. Hikobia 17: 315-320.

Hori K, Murakami N (2019) Origin of the Diplazium hachijoense complex (Athyriaceae). Phy-
toKeys 124: 57-76. https://doi.org/10.3897/phytokeys. 124.35242

IUCN (2012) Guidelines for Application of IUCN Red List Criteria at Regional and National
Levels: Version 4.0. Gland, Switzerland and Cambridge, UK.

82 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

Kato M (1973) Taxonomical evaluation of the articulated hairs found in the Athyriaceae. Acta
Phytotaxonomica et Geobotanica 25: 119-126.

Kato M (1977) Classification of Athyrium and allied genera of Japan. The Botanical Magazine
90(1): 23-40. https://doi.org/10.1007/BF02489467

Kato M (1984) A taxonomic study of the athyrioid fern genus Deparia with main reference to
the Pacific species. Journal of the Faculty of Science Section III 13: 371-430.

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGAX: Molecular evolutionary
genetics analysis across Computing Platforms. Molecular Biology and Evolution 35(6):
1547-1549. https://doi.org/10.1093/molbev/msy096

Kuo LY, Ebihara A, Shinohara W, Rouhan G, Wood KR, Wang CN, Chiou WL (2016) His-
torical biogeography of the fern genus Deparia (Athyriaceae) and its relation with poly-
ploidy. Molecular Phylogenetics and Evolution 104: 123-134. https://doi.org/10.1016/j.
ympev.2016.08.004 PubMed

Kuo LY, Ebihara A, Hsu TC, Rouhan G, Huang YM, Wang CN, Chiou WL, Kato M (2018)
Infrageneric revision of the fern genus Deparia (Athyriaceae, Aspleniineae, Polypodiales).
Systematic Botany 43(3): 645-655. https://doi.org/10.1600/036364418X697364

Kurita S (1963) Cytotaxonomical studies on some leptosporangiate ferns. Journal of the Col-
lege of Arts and Sciences. Chiba University 4: 43-52.

Li FW, Tan BC, Buchbender V, Moran RC, Rouhan G, Wang CN, Quandt D (2009) Iden-
tifying a mysterious aquatic fern gametophyte. Plant Systematics and Evolution 281(1):
77-86. https://doi.org/10.1007/s00606-009-0188-2

Mitui K (1966) Chromosome studies on Japanese ferns (2). Shokubutsu Kenkyu Zasshi 41: 60-64.

Mitui K (1968) Chromosomes and speciation in fern. Science Reports of the Tokyo Kyoiku
Daigaku. Section B 13: 285-333.

Mitui K (1970) Chromosomes studies on Japanese ferns (4). Shokubutsu Kenkyu Zasshi 45: 84-90.

Moran RC, Hanks JG, Sundue M (2019) Phylogenetic relationships of Neotropical lady ferns
(Athyriaceae), with a description of Ephemeropteris, gen. nov. Taxon 68(3): 425-441. htt-
ps://doi.org/10.1002/tax. 12063

Nakato N, Ebihara A (2018) Chromosome Numbers of Eleven Ferns in Japan. (Athyriaceae,
Dryopteridaceae and Tectariaceae). Bulletin of National Museum of Nature and Science,
Series B 44: 23-30.

Nakato N, Mitui K (1979) Intraspecific polyploidy in Diplazium subsinuatum (Wall.) Tagawa.
Jounal of Japanese Botany 54: 129-136.

Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press,
New York.

Otsuka K, Fujiwara M (1999) A new hybrid of the genus Deparia (Woodsiaceae) from Nagano
prefecture, Japan. The Journal of Phytogeography and Taxonomy 47: 107—110. [in Japanese]

PPG I (2016) A community-derived classification for extant lycophytes and ferns. Journal of
Systematics and Evolution 54(6): 563-603. https://doi.org/10.1111/jse.12229

Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarisation in
Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67(5): 901-904. https://doi.
org/10.1093/sysbio/syy032

Depariaxnanakuraensis K.Hori (Athyriaceae) 83

Ronquist F, Teslenko M, Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard
MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and
model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.
org/10.1093/sysbio/sys029

Rothfels CJ, Sundue MA, Kuo L-Y, Larsson A, Kato M, Schuettpelz E, Pryer KM (2012) A re-
vised family-level classification for eupolypod II ferns (Polypodiidae: Polypodiales). Taxon
61(3): 515-533. https://doi.org/10.1002/tax.613003

Sano R, Takamiya M, Kurita S, Ito M, Hasebe M (2000) Diplazium subsinuatum and Di.
tomitaroanum should be moved to Deparia according to molecular, morphological, and cy-
tological characters. Journal of Plant Research 113(2): 157-163. https://doi.org/10.1007/
PLO0013922

Serizawa S (1981) Notes of Japanese ferns (2). The Journal of Phytogeography and Taxonomy
29: 22-25. [in Japanese]

Shimura Y (1980) Notes of hybrid fern (2). The Journal of Phytogeography and Taxonomy 28:
1-42. [in Japanese]

Shinohara W, Takamiya M, Murakami N (2003) Taxonomic study of Japanese Deparia peterse-
nii (Woodsiaceae) based on cytological and molecular information. Acta Phytotaxonomica
et Geobotanica 54: 137-148.

Sundue MA, Rothfels CJ (2014) Stasis and convergence characterize morphological evolution in
eupolypod II ferns. Annals of Botany 113(1): 35-54. https://doi.org/10.1093/aob/mct247

Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three
noncoding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105-1109.
https://doi.org/10.1007/BF00037152

Appendix |

Voucher specimens for DNA analysis in this study. Data are in the order: Species
name —locality voucher (Herbarium); haplotype of plastid trnZ-F; allele of nuclear AKT.

Deparia xnanakuraensis K.Hori— JAPAN. Akita pref., Noshiro city, Futatsui
town, Nanakura-shrine, 23m alt., 40°12'9.48"N, 140°15'29.82"E, 7 Jul 2020, K. Hori
3391 (MAK, MBK); MT898446 (¢rnL-F); MT887301, MT887307 (AK1). ibid., K.
Hori 3392 (MAK, MBK); MT898447 (trnL-F); MT887302, MT887308 (AK1). ibid.,
K. Hori 3393 (MAK, MBK); MT898448 (trnL-F); MT887303, MT887309 (AK1).

D. pterorachis (Christ) M.Kato— JAPAN. Hokkaido Pref., Sapporo city, Minami-
ku, Jouzannkei, 530m alt., 42°55'36.8"N, 141°10'6.1"E, July 30 2018, K. Hori 3053
(MBK); MT898441 (¢rnL-F); MT887299 (AK1). ibid., Ebetsu city, Nopporo nature
park, July 30 2018, K. Hori 3054 (MBK); MT898442 (trnL-F); MT887300 (AK1).
ibid., K. Hori 3055 (MBK); MT898443 (trnL-F); LC421964 (AKI, Hori 2018).

D. viridifrons (Makino) M.Kato— JAPAN. Kochi pref., Takaoka county, Ochi
town, Mt. Yokogura, May 30 2018, K. Hori 2971 (MBK); LC421960 (érnL-F, Hori
and Murakami 2019); LC468191 (AKI, Hori 2018). ibid., Oct 17 2018, K. Hori

84 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020)

3060 (MAK); MT898444 (trnL-F); MT887305 (AK). ibid., Oct 17 2018, K. Hori
3061 (MAK); MT898445 (¢rnL-F); MT887306 (AKZ).

D. coreana (Christ) M.Kato— JAPAN. Aomori Pref., Kamikita county, Shichi-
nohe town, Jul 26 2018, Hori 3047 (MBK); MW051518 (tmL-F); MW051522,
MW051523 (AKI).

D. henryi (Baker) M.Kato— JAPAN. Kyoto Pref., Kyoto City, Jul 14 2018, Hori
3028 (MBK); MW051514 (zrnL-F); MW051527, MW0515278, MW051529 (AK7).

D. okuboana (Makino) M.Kato— JAPAN. Kyoto pref., Kyoto city, Jul 14 2018,
Hori 3033 (MBK); MW051515 (¢rnZ-F); MW051530, MW051531 (AK).

D. pycnosora (Christ) M.Kato var. albosquamata M.Kato— JAPAN. Nagano
Pref., Nagano city, Togakushi shrine, Okusha, Jul 9 2020, K. Hori 3382 (MAK);
MW051519 (trnL-F); MW051520, MW051521 (AKZ).

D. pycnosora (Christ) M. Kato var. mucilagina M.Kato— JAPAN. Nagano Pref.,
Nagano city, Togakushi shrine, Okusha, Jul 9 2020, K. Hori 3380 (MAK); MW051516
(trnL-F); MW051526 (AK).

D. pycnosora (Christ) M. Kato var. pycnosora M.Kato— JAPAN. Aomori Pref.,
Kamikita county, Touhoku town, Otsutomo, Jul 26 2018, K. Hori 3052 (MAK);
MW051517 (trnL-F); MW051524, MW051525 (AKI).

D. unifurcata (Baker) M.Kato— JAPAN. Kyoto Pref., Kyoto city, Jul 14 2018,
K. Hori 3029 (MBK); LC468192 (trnL-F, Hori and Murakami 2019); LC421961,
LC421962 (AKI, Hori 2018).